Patients with acute lung injury are placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may contribute to poor patient outcomes and worsening lung injury by generating large shear forces during repetitive reopening of fluid-filled alveoli and over-distending spared air-filled alveoli. Keratin intermediate filaments (IF) are the major structural proteins in alveolar epithelial cells that maintain the cell's mechanical integrity. Keratin IF undergo adaptive changes in response to shear stress. In preliminary studies we observed that the keratin IF network was disassembled in alveolar epithelial cells exposed to shear stress and that this was associated with alveolar epithelial cell injury. This application proposes to study the response of keratin IF to cyclic shear stress and stretch in alveolar epithelial cells utilizing cellular and molecular biology techniques via three interrelated specific aims: Specific Aim 1: To determine whether cyclic shear stress and/or stretch of alveolar epithelial cells causes keratin IF disassembly and degradation; Specific Aim 2: To determine whether cyclic shear stress and/or stretch causes degradation of keratin proteins occurs via the ubiquitin-proteasome pathway; Specific Aim 3: To identify the E2 and E3 enzymes involved in the ubiquitin-mediated degradation of alveolar epithelial cell keratin IF exposed to mechanical strain. Completion of these studies will provide new insights into the mechanisms responsible for ventilation induced mechanical injury to alveolar epithelial cells.